Multimodal Cardiac Imaging with gSTED

Imaging of heart sections was performed using a Leica TCS SP8 gated STED (gSTED) microscope, equipped with a white light laser and a 93x objective lens (HC PL APO CS2 93x GLYC, NA 1.30). For confocal images of mitochondria and DNA, Z-stacks in accordance with the Nyquist sampling criteria were taken by exciting the fluorophores at 488 nm and 594 nm, respectively, and Hybrid detectors collected fluorescent signals. Stimulated emission depletion of DNA channel was performed with a 775 nm depletion laser. 2D confocal and gSTED images were acquired sequentially with the optical zoom set to obtain a voxel size of 17 x 17 nm. Excitation was provided at 594 nm and Hybrid detectors collected signal. Gating between 0.3–6 ns was applied. Images were deconvolved with the Huygens software. Performance of the microscope and optimal depletion laser power were tested as previously described [65 (link)].